Transport domain of the erythrocyte anion exchange protein

Abstract

The anion transport domain of the anion exchange protein (AEP) of human erythrocyte membranes (band 3, 95 kD mol wt) was probed with the substrate and affinity label pyridoxal-5'-phosphate (PLP). Acting from outside, this probe labels two chymotryptic fragments of 65 and 35 kD of AEP but only the 35-kD fragment is protected from labeling by reversibly acting disulfonic stilbenes (DS). It is shown here by functional studies and by immunoblotting with anti-PLP antibodies that transmembrane gradients of anions determine the availability of a 35-kD fragment lys residue to surface labeling by PLP, in analogy with their effects on labeling of 65-kD fragment by DS. On this basis, it is suggested that both fragments contribute to the formation of the transport domain. However, unlike DS, PLP blocks transport when reacted from within released membranes, indicating that the 35-kD fragment might contain components of the mobile unit of the AEP. Using impermeant fluorescence quenchers of PLP of both complexation type (anti-PLP antibodies) or collisional type (acrylamide) as topological probes for PLP-labeled sites, it is deduced that the 65-kD PLP-labeled and the 35-kD PLP-labeled lys groups are inaccessible to macromolecules from either surface, but the 65-kD PLP-lys is accessible to low molecular weight molecules from without while the 35-kD PLP-labeled lys shows accessibility primarily from within the cell surface. The studies indicate that the accommodation of a wide class of anions by AEP might be associated with the flexibility of the transport domain of the protein and its capacity to undergo transport-related conformational changes.

Measurement of exofacially reactive lysines on human erythrocyte band 3 using pyridoxal 5′-phosphate

“…We believe that the difference in whole molecule PLP labeling stoichiometry between our study and the study published in this journal [1], arises from a substantial difference in labeling conditions. We added excess borohydride to mixtures of PLP and intact cells without significant dilution (<2%).…”

“…I read with interest the recent paper by Bar-Noy and Cabantchik [1] on pyridoxal 5'-phosphate (PLP) labeling of band 3. Their findings generally confirm our previous results [4] showing: (i) that both integral chymotryptic subdomains of band 3 (CH 17 and CH35) have Iysines which are "sensitive" to the DNDS (4,4'dinitrostilbene-2,2'-disulfonate)-induced conformational change [see 1, Note Added in Proof) and (ii) that exclusive (but partial) coverage of CH17 by PLP significantly inhibits anion transport under "symmetric" labeling conditions in chloride (see Fig.…”

“…We added excess borohydride to mixtures of PLP and intact cells without significant dilution (<2%). In contrast, Bar-Noy and Cabantchik [1] state that they added excess lysine (50 mM) and then washed their cells twice before adding the fixative. In contrast, Bar-Noy and Cabantchik [1] state that they added excess lysine (50 mM) and then washed their cells twice before adding the fixative.…”

The Band 3-Related AE Anion Exchanger Gene Family

“…These residues were selected because of their modeled position at the inside AE1 protein surface linearly adjacent to the putative final transmembrane loop in which resides the co valent binding site for pyridoxal-5'-phosphate, K869 [53]. Pyridoxal-5'-phosphate bound to K869 displays fluorescence quench ing behavior consistent with its exposure to both intracellular and extracellular solvent during the transport cycle [61].…”

The Cytoplasmic and Transmembrane Domains of AE2 Both Contribute to Regulation of Anion Exchange by pH

“…Alternatively, the sensor(s) might be located in a position that allows proton sensing of both intracellular and extracellular solutions, thus providing for dual sensing of pH i and pH o . Glu 681 (44) and Lys 851 (45) of human AE1 may be exposed to both sides of the red cell lipid bilayer. Such residues very likely contribute to the anion binding and translocation pathway through AE1.…”